Method for producing phosphorylated polysaccharide aqueous solution

Abstract

To provide a method for producing a phosphorylated polysaccharide aqueous solution enabling easy industrial production.SOLUTION: A method for producing a phosphorylated polysaccharide aqueous solution comprises reacting a mixed solution containing a polysaccharide, an alkali compound, and water with a phosphate ester compound at a temperature of 5-100°C.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a method for producing an aqueous solution of a phosphorylated polysaccharide. [Background technology]

[0002] Methods for producing aqueous solutions of phosphorylated polysaccharides have been investigated. Specifically, as shown in Patent Documents 1 and 2, for example, methods have been investigated in which a polysaccharide is reacted with monophosphoric acid or phosphorus oxychloride (POCl3) to obtain an aqueous solution of a phosphorylated polysaccharide. However, when an aqueous solution of phosphorylated polysaccharide is produced using monophosphoric acid, a reaction at high temperature is required, and caramelization and other reactions occur, making it difficult to control the reaction. On the other hand, POCl3 is highly decomposable and reactive in aqueous solution, so it must be reacted at low temperatures, and there is also the problem of generating hydrogen chloride gas. A method using phosphorus pentoxide is also known, but phosphorus pentoxide is highly hygroscopic and decomposes violently in water, so the phosphorylation efficiency is extremely poor in phosphorylation reactions in aqueous solutions. Therefore, the above-mentioned manufacturing method is difficult to use for industrial production. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] International Publication No. 2009 / 091001 [Patent Document 2] International Publication No. 2013 / 146669 Summary of the Invention [Problem to be solved by the invention]

[0004] The present invention aims to solve the above problems and to provide a method for producing an aqueous solution of phosphorylated polysaccharides that can be easily produced industrially. [Means for solving the problem]

[0005] In view of the above-mentioned problems, the present inventors have conducted studies and have found that by using a phosphate ester compound as a compound used for phosphorylation and by reacting the phosphate ester compound with a mixed solution containing a polysaccharide, an alkali compound, and water, the reaction can be sufficiently carried out without the need for a high temperature reaction, thereby solving the above-mentioned problems. Specifically, the above problems were solved by the following means. <1> A method for producing an aqueous phosphorylated polysaccharide solution, comprising reacting a mixed liquid containing a polysaccharide, an alkaline compound, and water with a phosphate ester compound at a temperature of 5 to 100°C. <2> The phosphate ester compound includes a compound represented by formula (P1): <1> A method for producing the aqueous phosphorylated polysaccharide solution described in [ka] (In formula (P1), R 1 , R 2 and R 3 are each independently a hydrogen atom or a substituent. 1 and R 3 , R 1 and R 2 , R 2 and R 3 , R 2 and R 2 may be bonded to each other to form a ring. 1 and R 2 is a hydroxyl group, and R 3 is not a hydrogen atom, and n is an integer from 1 to 200. <3> The phosphate ester compound has a structure represented by -POP-. <1> or <2> A method for producing the aqueous phosphorylated polysaccharide solution described in <4> The phosphate ester compound includes a cyclic phosphate ester compound. <1> ~ <3> 1. A method for producing the aqueous phosphorylated polysaccharide solution according to any one of the above. <5> mixing a polysaccharide, an alkaline compound, and water to obtain a mixed solution containing the polysaccharide, the alkaline compound, and water; <1> ~ <4> 1. A method for producing the aqueous phosphorylated polysaccharide solution according to any one of the above. <6> The temperature at which the polysaccharide, the alkaline compound, and the water are mixed is 5 to 100°C. <5> A method for producing the aqueous phosphorylated polysaccharide solution described in <7> In the mixed solution, the amount of polysaccharide is 0.1 to 2.5 parts by weight per 100 parts by weight of water. <5> or <6> A method for producing the aqueous phosphorylated polysaccharide solution described in <8> all steps from obtaining a mixed solution containing the polysaccharide, an alkaline compound, and water to reacting the mixed solution containing the polysaccharide, an alkaline compound, and water with a phosphate ester compound to obtain an aqueous phosphorylated polysaccharide solution are carried out at a temperature of 5 to 100°C; <5> ~ <7> 1. A method for producing the aqueous phosphorylated polysaccharide solution according to any one of the above. <9> the content of the alkaline compound in the mixed solution containing the polysaccharide, the alkaline compound, and water is 1 to 150 parts by weight relative to 100 parts by weight of the polysaccharide; <1> ~ <8> 1. A method for producing the aqueous phosphorylated polysaccharide solution according to any one of the above. <10> The polysaccharide comprises a water-soluble polysaccharide in which a total of two or more pentoses and / or hexoses are bonded via glycosidic bonds. <1> ~ <9> 1. A method for producing the aqueous phosphorylated polysaccharide solution according to any one of the above. <11> The functional group of the polysaccharide includes at least one selected from an alkyl group, a hydroxy group, and a carbonyl group. <1> ~ <10> 1. A method for producing the aqueous phosphorylated polysaccharide solution according to any one of the above. <12> The polysaccharide is a water-soluble polysaccharide in which a total of two or more pentoses and / or hexoses are bonded by glycosidic bonds, and the functional group of the polysaccharide includes at least one selected from an alkyl group, a hydroxyl group, and a carbonyl group. <1> ~ <11> 1. A method for producing the aqueous phosphorylated polysaccharide solution according to any one of the above. <13> The polysaccharide comprises at least one selected from the group consisting of pullulan, dextran, starch, amylose, guar gum, glucomannan, hydroxypropyl cellulose, carboxymethyl cellulose, agarose, carrageenan, lactose, sucrose, sucralose, cellobiose, trehalose, maltose, isomaltulose, maltotriose, maltodextrin, cyclodextrin, glycosylsucrose, cycloamylose, glycogen, cluster dextrin, and lentinan; <1> ~ <12> 1. A method for producing the aqueous phosphorylated polysaccharide solution according to any one of the above. <14> the phosphate ester compound comprises a compound represented by formula (P1), The phosphate ester compound includes a cyclic phosphate ester compound. <1> ~ <13> 1. A method for producing the aqueous phosphorylated polysaccharide solution according to any one of the above. [ka] (In formula (P1), R 1 , R 2 and R 3 are each independently a hydrogen atom or a substituent. 1 and R 3 , R 1 and R 2 , R 2 and R 3 , R 2 and R 2 may be bonded to each other to form a ring. 1 and R 2 is a hydroxyl group, and R 3 is not a hydrogen atom, and n is an integer from 1 to 200. <15> and reacting the mixture with a phosphate ester compound, and then desalting the resulting reaction solution with an ultrafiltration membrane to remove by-products. <1> ~ <14> 1. A method for producing the aqueous phosphorylated polysaccharide solution according to any one of the above. <16> The purified product from which the by-products have been removed is subjected to cross-flow filtration using an ultrafiltration membrane to concentrate the phosphorylated polysaccharide to 0.15 to 10 wt %. <15> A method for producing the aqueous phosphorylated polysaccharide solution described in <17> <1> ~ <16> 1. A phosphorylated polysaccharide aqueous solution obtained by the method for producing a phosphorylated polysaccharide aqueous solution according to any one of the above. [Effects of the Invention]

[0006] The present invention makes it possible to provide a method for producing an aqueous solution of phosphorylated polysaccharides that can be easily produced industrially. DETAILED DESCRIPTION OF THE INVENTION

[0007] Hereinafter, an embodiment for carrying out the present invention (hereinafter simply referred to as "the present embodiment") will be described in detail. Note that the present embodiment is an example for explaining the present invention, and the present invention is not limited to only this embodiment. In this specification, the symbol "to" is used to mean that the numerical values ​​before and after it are included as the upper and lower limits. Furthermore, any combination of the upper and lower limit values ​​of the numerical values ​​in this specification is an example of this embodiment. In this specification, various physical properties and characteristic values ​​are those at 25°C unless otherwise specified. In the description of groups (atomic groups) in this specification, when a notation does not specify whether they are substituted or unsubstituted, it includes both groups (atomic groups) that have no substituents and groups (atomic groups) that have substituents. For example, the term "alkyl group" includes not only alkyl groups that have no substituents (unsubstituted alkyl groups) but also alkyl groups that have substituents (substituted alkyl groups). In this specification, when a notation does not specify whether they are substituted or unsubstituted, it is preferable that they be unsubstituted.

[0008] In this specification, the term "step" refers not only to an independent step, but also to a step that cannot be clearly distinguished from other steps, as long as the intended effect of the step is achieved.

[0009] In this specification, the reaction of a mixed liquid containing a polysaccharide, an alkaline compound, and water with a phosphate ester compound is referred to as a "phosphorylation reaction."

[0010] The method for producing a phosphorylated polysaccharide aqueous solution of the present embodiment is characterized by comprising reacting a mixed solution containing a polysaccharide, an alkali compound, and water with a phosphate ester compound at a temperature of 5 to 100° C. By adopting such a configuration, it is possible to provide a method for producing a phosphorylated polysaccharide aqueous solution that can be easily produced industrially. In particular, an aqueous solution of phosphorylated polysaccharide can be easily obtained without cooling or heating. Furthermore, caramelization of the resulting phosphorylated polysaccharide can be effectively suppressed. Furthermore, it becomes possible to control the molecular weight of the resulting phosphorylated polysaccharide. Furthermore, it becomes possible to control the phosphorus content of the resulting phosphorylated polysaccharide. It is also highly valuable in that aqueous solutions of phosphorylated polysaccharides can be produced industrially.

[0011] That is, when a polysaccharide mixture solution is phosphorylated using POCl3, energy is required for cooling, etc., due to the high reactivity of POCl3. In this embodiment, an aqueous polysaccharide phosphate solution can be produced by a mild reaction.

[0012] In the method for producing a phosphorylated polysaccharide aqueous solution of this embodiment, a mixed solution containing a polysaccharide, an alkaline compound, and water is used. As long as the polysaccharide, alkaline compound, and water are mixed, the polysaccharide does not need to be dissolved in water or the alkaline compound. Preferably, the aqueous solution is one in which the polysaccharide is dissolved in water at 25°C. The mixed solution used in this embodiment can be produced by mixing a polysaccharide, an alkaline compound, and water to obtain a mixed solution containing the polysaccharide, the alkaline compound, and water. Alternatively, a commercially available mixed solution containing the polysaccharide, the alkaline compound, and water may be used. In this embodiment, the temperature at which the polysaccharide, alkaline compound, and water are mixed is preferably 5°C or higher, more preferably 8°C or higher, even more preferably 10°C or higher, even more preferably 15°C or higher, and even more preferably 20°C or higher, and is preferably 100°C or lower, more preferably 80°C or lower, even more preferably 60°C or lower, even more preferably 50°C or lower, and even more preferably 40°C or lower. By setting the temperature at or above the lower limit, the solubility of the polysaccharide tends to be further improved. On the other hand, by setting the temperature at or below the upper limit, caramelization of the sugar and a decrease in molecular weight can be suppressed, and the energy load for temperature adjustment tends to be reduced. In this embodiment, the polysaccharide, the alkaline compound, and the water may be mixed without heating or cooling. Here, heating and cooling means that the reaction system is not actively heated or cooled, and does not include an unintentional increase in the temperature of the reaction system due to the operation of a mechanical device or the like. The reaction system in this embodiment refers to a reaction system (phosphorylation reaction system) in which a mixed solution containing a polysaccharide, an alkali compound, and water is reacted with a phosphate ester compound at a temperature of 5 to 100° C. The reaction system in this embodiment typically contains a polysaccharide, an alkali compound, water, a phosphate ester compound, and reaction products or intermediates thereof, and the total of the polysaccharide, alkali compound, water, phosphate ester compound, and reaction products or intermediates thereof preferably accounts for 80% by weight or more of the reaction system, more preferably 90% by weight or more, even more preferably 95% by weight or more, and even more preferably 99% by weight or more.

[0013] The aqueous solution of this embodiment preferably has a pH greater than 7, more preferably 8 or greater, even more preferably 9 or greater, even more preferably 10 or greater, even more preferably 11 or greater, and preferably 14 or less, more preferably less than 14, even more preferably 13.5 or less, even more preferably 13 or less. By setting the pH at or above the lower limit, the nucleophilicity of the polysaccharide tends to increase, and the efficiency of the phosphorylation reaction tends to increase. Furthermore, by setting the pH at or below the upper limit, decomposition of the phosphate ester compound tends to be suppressed.

[0014] In this embodiment, the amount of polysaccharide relative to 100 parts by weight of water in the mixed solution is preferably 0.1 parts by weight or more, more preferably 0.2 parts by weight or more, even more preferably 0.3 parts by weight or more, and even more preferably 0.4 parts by weight or more, and is preferably 2.5 parts by weight or less, more preferably 2.0 parts by weight or less, and even more preferably 1.5 parts by weight or less. When a higher phosphorus content is desired, it is more preferably 1.0 part by weight or less, and even more preferably 0.8 parts by weight or less. By setting the amount at or above the lower limit, the yield per unit amount of reaction solution tends to increase, and production efficiency tends to improve. Furthermore, by setting the amount at or below the upper limit, high viscosity and gelation of the aqueous solution tend to be suppressed. By reducing the proportion of polysaccharide in the mixed solution, the phosphorus content can be increased, and the phosphorus content of the resulting phosphorylated polysaccharide can be easily controlled. In this embodiment, only one type of polysaccharide may be used, or two or more types may be used. When two or more types are used, it is preferable that the total amount is within the above range.

[0015] In this embodiment, the amount of alkali compound in the mixed solution per 100 parts by weight of polysaccharide is preferably 1 part by weight or more, more preferably 2 parts by weight or more, and depending on the intended use, is even more preferably 5 parts by weight or more, even more preferably 10 parts by weight or more, even more preferably 50 parts by weight or more, even more preferably 80 parts by weight or more, and is preferably 150 parts by weight or less, more preferably 140 parts by weight or less. By setting the amount at or above the lower limit, the nucleophilicity of the polysaccharide tends to be improved, and the efficiency of the phosphorylation reaction tends to be further improved. On the other hand, by setting the amount at or below the upper limit, decomposition of the phosphate ester compound tends to be more effectively suppressed. In this embodiment, only one type of alkaline compound may be used, or two or more types may be used. When two or more types are used, the total amount is preferably within the above range.

[0016] The type of polysaccharide used in this embodiment is not particularly limited, and may be either a water-soluble or water-insoluble polysaccharide. Preferably, the polysaccharide is a water-soluble polysaccharide that dissolves in water at 25°C. The polysaccharide used in this embodiment preferably contains a water-soluble polysaccharide in which a total of two or more five-carbon sugars (pentoses) and / or six-carbon sugars (hexoses) are linked by glycosidic bonds, and more preferably contains a water-soluble polysaccharide in which a total of two or more six-carbon sugars are linked by glycosidic bonds. The number of pentoses and / or hexoses constituting the polysaccharide is preferably 2 or more, more preferably 5 or more, and is preferably 3000 or less, more preferably 2500 or less. The glycosidic bonds are preferably α-1,4 glycosidic bonds and / or α-1,6 glycosidic bonds, and it is more preferable that monosaccharides are linked by α-1,4 glycosidic bonds and α-1,6 glycosidic bonds. The polysaccharide used in this embodiment preferably has functional groups that include at least one selected from an alkyl group, a hydroxy group, and a carbonyl group, and more preferably has functional groups that consist of only one or more selected from an alkyl group, a hydroxy group, and a carbonyl group. The polysaccharide used in this embodiment preferably has at least one or more functional groups selected from the group consisting of a methyl group, a hydroxy group, and a methylhydroxy group bonded to a pentose and / or a hexose.

[0017] The weight-average molecular weight of the polysaccharide is preferably 300 Da or more, more preferably 5000 Da or more, even more preferably 10 kDa or more, even more preferably 100 kDa or more, and even more preferably 200 kDa or more, and is preferably 1000 kDa or less, more preferably 900 kDa or less, even more preferably 850 kDa or less, even more preferably 800 kDa or less, and even more preferably 700 kDa or less. When the mixed solution used in this embodiment contains two or more types of polysaccharides, the molecular weight is the molecular weight of the mixture of polysaccharides. The molecular weight of the polysaccharides is determined according to gel permeation chromatography GPC.

[0018] Specific examples of polysaccharides include pullulan, dextran, starch, amylose, guar gum, glucomannan, hydroxypropyl cellulose, carboxymethyl cellulose, agarose, carrageenan, lactose, sucrose, sucralose, cellobiose, trehalose, maltose, isomaltulose, maltotriose, maltodextrin, cyclodextrin, glycosylsucrose, cycloamylose, glycogen, cluster dextrin, and lentinan, with pullulan being preferred.

[0019] The type of alkaline compound used in this embodiment is not particularly limited, but it is preferable that the pH of the mixed solution can be adjusted to a desired value. The alkali compound used in this embodiment may be an inorganic compound or an organic compound, but is preferably an inorganic compound, more preferably an alkali hydroxide compound, further preferably an alkali metal hydroxide or an alkaline earth metal hydroxide, still more preferably sodium hydroxide, potassium hydroxide, calcium hydroxide, lithium hydroxide, or magnesium hydroxide, and still more preferably sodium hydroxide. In addition, as the alkaline compound, sodium methoxide, sodium ethoxide, potassium methoxide, potassium ethoxide, sodium phosphate, potassium phosphate, sodium silicate, potassium silicate, etc. can also be used.

[0020] In this embodiment, a phosphate ester compound is used for phosphorylating polysaccharides. By using a phosphate ester compound, the phosphorylation reaction can be easily carried out without heating or cooling, and an aqueous solution of phosphorylated polysaccharides can be easily obtained. Until now, it has been difficult to obtain an aqueous solution of phosphorylated polysaccharides under mild reaction conditions, but in this embodiment, an aqueous solution of phosphorylated polysaccharides can be obtained even under mild conditions. The phosphate ester compound used in this embodiment preferably includes a compound represented by formula (P1). [ka] (In formula (P1), R 1 , R 2 and R 3 are each independently a hydrogen atom or a substituent. 1 and R 3 , R 1 and R 2 , R 2 and R 3 , R 2 and R 2 may be bonded to each other to form a ring. 1 and R 2 is a hydroxyl group, and R 3 is not a hydrogen atom, and n is an integer from 1 to 200.

[0021] In formula (P1), R 1 and R 3 , R 1 and R 2 , R 2 and R 3 , R 2 and R 2 When one or more of the above are bonded to each other to form a ring, they may be a single bond, and a single bond is preferred.

[0022] R 1 , R 2 and R 3 are substituents, each independently represents -O - M m+ (M is an alkali metal or an alkaline earth metal. When M is an alkali metal, m is 1. When M is an alkaline earth metal, m is 2. When m is 2, there are two molecules and two -O - and is paired with. ), a halogen atom, a cyano group, a nitro group, a hydroxy group, an alkyl group, an alkoxy group, an aryl group, an aryloxy group, a heterocyclic group, a heterocyclicoxy group, an alkenyl group, an alkylsulfanyl group, an arylsulfanyl group, an acyl group, or an amino group is preferred, a halogen atom, an alkyl group, an alkoxy group, an aryl group, an aryloxy group, an alkenyl group, or an acyl group is more preferred, an alkyl group, an aryl group, an alkoxy group, or an alkenyl group is even more preferred, an alkyl group or an alkoxy group is even more preferred, and an alkyl group is even more preferred. The alkali metal is preferably lithium, sodium or potassium, more preferably sodium or potassium, and even more preferably sodium. The alkaline earth metal is preferably beryllium, magnesium, calcium, strontium, or barium, and more preferably magnesium or calcium. Preferably, M is an alkali metal. n is an integer of 1 to 200, preferably 2 or more, more preferably 3 or more, and may be 150 or less, 100 or less, 50 or less, 30 or less, 20 or less, 10 or less, 8 or less, or 5 or less depending on the application, etc.

[0023] Formula (P1) is preferably a compound represented by formula (P1-1). [ka] (In formula (P1-1), R 1 and R 3 are each independently a hydrogen atom or a substituent. 1 and R 3 may be bonded to each other to form a ring. n is an integer of 1 to 200. M A are each independently an alkali metal.)

[0024] In formula (P1-1), R 1 , R 3 , and n is R in formula (P1) 1 , R 3 The same definitions and preferred ranges are also given for M. A + is preferably a sodium ion or a potassium ion.

[0025] Formula (P1) is preferably a compound represented by formula (P1-2). [ka] (In formula (P1-2), n is an integer of 1 to 200. M A are each independently an alkali metal.)

[0026] In formula (P1-2), n has the same meaning as n in formula (P1), and the preferred range is also the same. A + is preferably a sodium ion or a potassium ion.

[0027] The phosphate compound may be a phosphate salt, such as a sodium salt, a potassium salt, a calcium salt, or a magnesium salt, with sodium and potassium salts being preferred, and sodium salts being more preferred.

[0028] Furthermore, the phosphate ester compound preferably has a structure represented by -POP-. An example of a compound having a structure represented by -POP- is a compound represented by formula (P1). In a compound represented by -POP-, a hydrogen atom or a substituent is usually bonded to the P atom. The phosphate ester compound preferably includes a cyclic phosphate ester compound. An example of the cyclic phosphate ester compound is a compound represented by formula (P1-2).

[0029] The phosphate ester compound used in this embodiment may be solid or liquid when added to the reaction system, but is preferably solid, and more preferably powder. By using a powder, prior dissolution is not required compared to an aqueous solution, and the number of operations during production can be reduced. On the other hand, the phosphate ester compound may be liquid when added. The liquid may be an aqueous solution of the phosphate ester compound. Using an aqueous solution tends to be easier to handle than a powder.

[0030] Specific examples of the phosphate ester compound include sodium trimetaphosphate, phenyl dichlorophosphate, diphenyl chlorophosphate, and sodium hexametaphosphate.

[0031] The amount of the phosphate ester compound is preferably 10 parts by weight or more, more preferably 50 parts by weight or more, even more preferably 75 parts by weight or more, even more preferably 100 parts by weight or more, and even more preferably 120 parts by weight or more, relative to 100 parts by weight of the polysaccharide, and is preferably 5,000 parts by weight or less, more preferably 3,000 parts by weight or less, even more preferably 2,000 parts by weight or less, even more preferably 1,000 parts by weight or less, and even more preferably 600 parts by weight or less. By adjusting the amount to be equal to or greater than the lower limit, the phosphorus content of the resulting phosphorylated polysaccharide tends to be increased. By adjusting the amount to be equal to or less than the upper limit, purification becomes easier and the amount of phosphorus compounds in the filtrate during desalting decreases, which tends to reduce the cost of recovery, reuse, or disposal.

[0032] The method for producing a phosphorylated polysaccharide aqueous solution of this embodiment comprises reacting a mixed liquid containing a polysaccharide, an alkaline compound, and water with a phosphate ester compound at a temperature of 5 to 100°C. An example of the phosphorylation reaction of this embodiment is shown below. [ka]

[0033] In this embodiment, the phosphorylation reaction can proceed at 5 to 100° C., making industrial production possible. The temperature of the phosphorylation reaction is 5°C or higher, preferably 10°C or higher, more preferably 11°C or higher, even more preferably 15°C or higher, even more preferably 18°C ​​or higher, even more preferably 20°C or higher, and preferably 80°C or lower, more preferably 65°C or lower, and even more preferably 60°C or lower. By setting the temperature at or above the lower limit, phosphorylation tends to proceed efficiently. By setting the temperature at or below the upper limit, decomposition of the phosphorylated polysaccharide tends to be suppressed. Furthermore, by adjusting the temperature of the phosphorylation reaction, it becomes possible to precisely control the molecular weight of the resulting phosphorylated polysaccharide. The phosphorylation reaction of this embodiment may be carried out without heating or cooling. Here, heating and cooling mean that the reaction system is not actively heated or cooled, and does not include unintentional increases in the temperature of the reaction system due to the operation of a mechanical device or the like.

[0034] The reaction time for the phosphorylation reaction in this embodiment is preferably 10 minutes or more, more preferably 30 minutes or more, even more preferably 1 hour or more, still more preferably 3 hours or more, and even more preferably 5 hours or more, and is preferably 30 hours or less, more preferably 20 hours or less, even more preferably 15 hours or less, still more preferably 10 hours or less, and even more preferably 7 hours or less. By setting the reaction time at or above the lower limit, the phosphorus content of the product tends to increase. Furthermore, by setting the reaction time at or below the upper limit, production efficiency tends to be further improved.

[0035] In this embodiment, as described above, the temperature at which the polysaccharide, alkaline compound, and water are mixed is preferably 5 to 100° C. Furthermore, it is preferable that all steps from obtaining a mixed solution containing the polysaccharide, alkaline compound, and water to reacting the mixed solution containing the polysaccharide, alkaline compound, and water with a phosphate ester compound to obtain an aqueous phosphorylated polysaccharide solution are carried out at a temperature of 5 to 100° C. By adopting such a configuration, caramelization of the resulting phosphorylated polysaccharide can be effectively suppressed. In this embodiment, the temperature of the entire reaction system from obtaining the mixed solution to reacting the mixed solution with a phosphate ester compound to obtain a phosphorylated polysaccharide aqueous solution is preferably 10°C or higher, more preferably 11°C or higher, even more preferably 15°C or higher, even more preferably 18°C ​​or higher, and even more preferably 20°C or higher, and is preferably 80°C or lower, more preferably 65°C or lower, even more preferably 50°C or lower, even more preferably 45°C or lower, and even more preferably 40°C or lower. By setting the temperature at or above the lower limit, the solubility of the polysaccharide tends to be further improved. On the other hand, by setting the temperature at or below the upper limit, caramelization of the sugar and a decrease in molecular weight can be suppressed, and the energy load for temperature adjustment tends to be reduced.

[0036] In this embodiment, the molecular weight of the resulting phosphorylated polysaccharide can be adjusted by controlling the temperature of the phosphorylation reaction, i.e., the decomposition of the polysaccharide main chain can be precisely controlled. In this embodiment, for example, when pullulan is used as the polysaccharide and trimetaphosphoric acid is used as the phosphorylating agent, the phosphorylation reaction is carried out at 5 to 35°C, and the main chain of the polysaccharide is decomposed to obtain a phosphorylated polysaccharide having a weight-average molecular weight of 200 to 300 kDa. In this embodiment, for example, when pullulan is used as the polysaccharide, the phosphorylation reaction is carried out at 35 to 45°C, and the main chain of the polysaccharide is decomposed to obtain a phosphorylated polysaccharide having a weight-average molecular weight of 100 to 200 kDa. In this embodiment, for example, when pullulan is used as the polysaccharide, the phosphorylation reaction is carried out at 45 to 60°C, and the main chain of the polysaccharide is decomposed to obtain a phosphorylated polysaccharide having a weight-average molecular weight of 50 to 100 kDa. In this embodiment, for example, when pullulan is used as the polysaccharide, the phosphorylation reaction is carried out at 60 to 100°C, and the main chain of the polysaccharide is decomposed to obtain a phosphorylated polysaccharide having a weight-average molecular weight of 1 to 50 kDa.

[0037] The method for producing the phosphorylated polysaccharide aqueous solution of this embodiment may be a batch method or a flow method (continuous method). Examples of methods for producing an aqueous phosphorylated polysaccharide solution include a method in which a phosphate ester compound is added to a reaction system all at once, a method in which a phosphate ester compound is continuously added to a reaction system, and a method in which a phosphate ester compound is added to a reaction system in divided portions multiple times. Of these, the method in which a phosphate ester compound is added to a reaction system in divided portions multiple times is preferred.

[0038] When the phosphate ester compound is added to the reaction system in multiple batches, the amount of the phosphate ester compound added to the reaction system in each batch is preferably 0.001% by weight or more, more preferably 0.01% by weight or more, even more preferably 0.1% by weight or more, and even more preferably 0.2% by weight or more, and is preferably 1% by weight or less, and more preferably 0.5% by weight or less, relative to the total amount of the reaction solution at the time of addition.

[0039] When the phosphate ester compound is continuously supplied to the reaction system, it is preferable to supply an aqueous solution of the phosphate ester compound to the reaction system at a rate of 0.2 to 10 parts by weight per minute relative to 100 parts by weight of the raw polysaccharide.

[0040] Examples of phosphorylated polysaccharides obtainable by the phosphorylated polysaccharide production method of this embodiment are shown below, but it goes without saying that the present invention is not limited to these. [ka] In the above, P X are each independently preferably the following structure: and n is a number from 1 to 1,000. [ka] The * denotes a binding site to the polysaccharide. Each X is independently a sodium atom, a potassium atom, or a hydrogen atom, preferably a sodium atom. Alternatively, the X may be a mixture of two or more of these.

[0041] In the phosphorylated polysaccharide obtained by the method for producing a phosphorylated polysaccharide of this embodiment, the hydroxyl groups of the constituent monosaccharides are usually phosphorylated, but it is preferable that the 6-position of the constituent monosaccharides is phosphorylated.

[0042] The phosphorus content of the phosphorylated polysaccharide obtained by the phosphorylated polysaccharide production method of this embodiment is preferably 0.1% by weight or more, more preferably 0.5% by weight or more, even more preferably 1% by weight or more, even more preferably 2% by weight or more, even more preferably 3% by weight or more, and preferably 10% by weight or less.

[0043] In this embodiment, after the phosphorylation reaction, the product may be used as is or may be purified. Specifically, it is preferable that after the mixed solution is reacted with the phosphate ester compound, the resulting reaction solution is desalted using an ultrafiltration membrane to remove by-products. It is also preferable to concentrate the purified product from which by-products have been removed to a phosphorylated polysaccharide content of 0.15 to 10% by weight by cross-flow filtration using an ultrafiltration membrane.

[0044] The phosphorylated polysaccharide obtained by the production method of this embodiment has excellent bioaffinity and bioabsorbability, as well as excellent adhesiveness in an aqueous environment. Furthermore, when mixed with a bioactive drug, it has excellent drug-carrying and sustained-release capabilities. [Example]

[0045] The present invention will be explained in more detail below with reference to examples. The materials, amounts used, ratios, processing details, processing procedures, etc. shown in the following examples can be appropriately changed without departing from the spirit of the present invention. Therefore, the scope of the present invention is not limited to the specific examples shown below. If the measuring instruments used in the examples are difficult to obtain due to discontinuation or the like, measurements can be made using other instruments with equivalent performance.

[0046] Example 1 5 g of sodium hydroxide and 5 g of pullulan (Hayashibara Co., Ltd., weight-average molecular weight approximately 300 kDa) were dissolved in 375 mL of water and stirred overnight at room temperature (25 °C). 6.7 g of sodium trimetaphosphate was then added, and the resulting reaction solution was stirred for an additional 6 hours at room temperature. The resulting solution was desalted by cross-flow filtration using an ultrafiltration membrane with a molecular weight cutoff of 5000 until the electrical conductivity of the filtrate reached 40 μS / cm or less. The solution was then concentrated by cross-flow filtration to a phosphorylated polysaccharide concentration of approximately 4 wt%, and the solvent was removed using a rotary evaporator. The solution was then dried in a vacuum dryer for 48 hours to completely remove the solvent, yielding phosphorylated pullulan. The resulting phosphorylated pullulan had a weight-average molecular weight of approximately 200 kDa and a phosphorus content of 1.4 wt%. The phosphorylated polysaccharide aqueous solution was easily obtained under mild conditions.

[0047] Examples 2 to 5 In Example 1, the amount of polysaccharide relative to water, the amount of alkaline compound relative to polysaccharide, the amount of phosphorus compound relative to polysaccharide, and the temperature of the phosphorylation reaction were changed as shown in Table 1, but the rest was the same. In all of Examples 2 to 5, aqueous solutions of phosphorylated polysaccharides were easily obtained under mild conditions.

[0048] (Examples 6 and 7) In Examples 1 and 4, the type of polysaccharide was changed from pullulan to dextran (manufactured by Sigma-Aldrich) as shown in Table 2, but the other procedures were the same.

[0049] Example 8 1 g of sodium hydroxide and 1 g of pullulan (Hayashibara Co., Ltd., weight-average molecular weight approximately 300 kDa) were dissolved in 75 mL of water, and the mixture was stirred overnight at room temperature (25°C). 1.18 g of diphenyl chlorophosphate (Tokyo Chemical Industry Co., Ltd.) was then added, yielding a mixture in which the phosphorylating agent was dispersed as fine droplets. This mixture was stirred at room temperature for 30 hours, yielding a homogeneous reaction solution. The resulting reaction solution was treated in the same manner as in Example 1.

[0050] (Comparative Example 1) As shown in Table 3, the procedure of Example 1 was repeated except that sodium trimetaphosphate was replaced with monophosphoric acid (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), but almost no phosphorylated polysaccharide was obtained.

[0051] (Comparative Example 2) In Comparative Example 1, when the temperature of the phosphorylation reaction was changed to 170°C, phosphorylated polysaccharide was obtained, as shown in Table 3. This indicates that heating is necessary to obtain phosphorylated polysaccharide. Furthermore, the obtained phosphorylated polysaccharide was brown in color.

[0052] (Comparative Example 3) In Example 1, as shown in Table 3, sodium trimetaphosphate was replaced with phosphorus oxychloride (POCl3) (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), but the other steps were carried out in the same manner. However, hydrogen chloride was generated, creating a dangerous situation.

[0053] Comparative Example 4 In Comparative Example 3, when the temperature of the phosphorylation reaction was changed to 0°C, phosphorylated polysaccharide was obtained, as shown in Table 3. In other words, it was found that cooling was necessary to obtain phosphorylated polysaccharide.

[0054] [Table 1]

[0055] [Table 2]

[0056] [Table 3]

[0057] (Coloring of phosphorylated polysaccharides) The presence or absence of coloration of the resulting phosphorylated polysaccharide was visually judged by five experts and determined by majority vote.

[0058] (Molecular weight adjustment) From Example 1, it was confirmed that phosphorylated pullulan having a weight-average molecular weight of 200 to 300 kDa can be obtained by carrying out the phosphorylation reaction at 0 to 35° C. and decomposing the main chain of pullulan. In Example 5, it was confirmed that phosphorylated pullulan having a weight-average molecular weight of 1 to 50 kDa can be obtained by carrying out the phosphorylation reaction at 60 to 100° C. and decomposing the main chain of pullulan.

[0059] (Measurement of molecular weight of phosphorylated polysaccharides) The molecular weight was measured by GPC analysis (column: two columns, Shodex OHpak SB-804HQ and SB-805HQ, connected together, mobile phase: 0.2 M aqueous sodium nitrate solution). The resulting phosphorylated polysaccharide was dissolved in the mobile phase to obtain a 2 mg / mL sample solution, which was then filtered through a syringe filter (hydrophilic PTFE, pore size: 0.20 μm, manufactured by Advantec) and subjected to GPC analysis.

[0060] (phosphorus content) The resulting phosphorylated polysaccharide was dissolved in water to prepare a 0.5 wt% aqueous solution, which was then further diluted 200 times with water to obtain a 25 ppm phosphorylated polysaccharide aqueous solution. The phosphorus content was calculated using a commercially available total phosphorus measurement kit (manufactured by Kyoritsu Chemical Research Institute). According to the kit instructions, 1 mL of sample solution was placed in a pressure-resistant bottle, and one drop of R1 reagent containing sulfuric acid and 0.5 mL of R2 reagent containing potassium peroxodisulfate were added. The sample bottle was heated for over 30 minutes using the attached high-pressure decomposer to decompose the phosphorus compounds in the sample into phosphate ions. The resulting phosphate ions were measured using molybdenum blue spectrophotometry to determine the total phosphorus concentration in the sample solution. The phosphorus content in the phosphorylated polysaccharide was calculated from the measured total phosphorus concentration and the concentration of phosphorylated polysaccharide in the sample solution.

[0061] (Adjustment of phosphorus content) Whether or not the phosphorus content can be adjusted by changing the reaction conditions was determined by comparing with the phosphorus content in Example 1. Examples and comparative examples marked with "-" were not evaluated. As is clear from Example 2, the phosphorus content could be increased by reducing the amount of polysaccharide relative to water. As is clear from Example 3, the phosphorus content could be reduced by reducing the amount of alkaline compound. As is clear from Example 5, the phosphorus content could be increased by increasing the amount of phosphate relative to the polysaccharide.

[0062] (Comparative Example 5) The examples of Patent No. 557960 were reproduced. Polysaccharide-based scaffolds were prepared using a 75:25 mixture of pullulan and dextran (pullulan, weight-average molecular weight 200,000, Hayashibara Inc., Okayama, Japan; dextran, weight-average molecular weight 500,000, Pharmacia). A polysaccharide solution was prepared by dissolving 9 g of pullulan and 3 g of dextran in 40 mL of distilled water. Sodium carbonate (8 g) was then added to the polysaccharide solution, and stirring was continued until a homogeneous mixture was obtained. The polysaccharide was chemically crosslinked using the crosslinker trisodium trimetaphosphate (STMP) (Sigma, St. Louis) under alkaline conditions. Briefly, 1 mL of 10 M sodium hydroxide was added to 10 g of the polysaccharide solution, followed by 1 mL of water containing 300 mg of STMP. The mixture was then poured into a Petri dish (Nunclon®, #150288) and incubated at 50°C for 15 minutes. The resulting hydrogel was immediately immersed in a large beaker containing 20% ​​acetic acid solution for at least 30 minutes. The resulting scaffold was washed extensively with phosphate-buffered saline (pH 7.4) and then with distilled water for at least two days. After the freeze-drying step, the porous scaffold was stored at room temperature (25°C) until use. A gel-like consistency was obtained, and no aqueous solution was obtained.

Claims

1. The method comprises reacting a mixed liquid containing a polysaccharide, an alkaline compound, and water with a phosphoric acid ester compound at a temperature of 5 to 100°C. A method for producing an aqueous solution of phosphorylated polysaccharides.

2. The method for producing a phosphorylated polysaccharide aqueous solution according to claim 1 , wherein the phosphate ester compound comprises a compound represented by formula (P1): 【Chemistry 1】 (In formula (P1), R １ , R ２ and R ３ are each independently a hydrogen atom or a substituent. １ and R ３ , R １ and R ２ , R ２ and R ３ , R ２ and R ２ may be bonded to each other to form a ring. １ and R ２ is a hydroxyl group, and R ３ is not a hydrogen atom, and n is an integer of 1 to 200.

3. 3. The method for producing a phosphorylated polysaccharide aqueous solution according to claim 1, wherein the phosphate ester compound has a structure represented by -P-O-P-.

4. The method for producing a phosphorylated polysaccharide aqueous solution according to claim 1 or 2, wherein the phosphate ester compound comprises a cyclic phosphate ester compound.

5. 3. A method for producing the phosphorylated polysaccharide aqueous solution according to claim 1, comprising mixing a polysaccharide, an alkaline compound, and water to obtain a mixed solution containing the polysaccharide, the alkaline compound, and water.

6. The method for producing a phosphorylated polysaccharide aqueous solution according to claim 5, wherein the polysaccharide, the alkaline compound and the water are mixed at a temperature of 5 to 100°C.

7. 6. The method for producing a phosphorylated polysaccharide aqueous solution according to claim 5, wherein the amount of the polysaccharide in the mixture is 0.1 to 2.5 parts by weight per 100 parts by weight of water.

8. 6. The method for producing a phosphorylated polysaccharide aqueous solution according to claim 5, wherein all steps from obtaining the mixed solution containing the polysaccharide, the alkaline compound, and water to reacting the mixed solution containing the polysaccharide, the alkaline compound, and water with a phosphate ester compound to obtain the phosphorylated polysaccharide aqueous solution are carried out at a temperature of 5 to 100°C.

9. 3. The method for producing a phosphorylated polysaccharide aqueous solution according to claim 1, wherein the content of the alkaline compound in the mixed solution containing the polysaccharide, the alkaline compound, and water is 1 to 150 parts by weight per 100 parts by weight of the polysaccharide.

10. The method for producing a phosphorylated polysaccharide aqueous solution according to claim 1 or 2, wherein the polysaccharide comprises a water-soluble polysaccharide in which a total of two or more pentoses and / or hexoses are bonded via glycosidic bonds.

11. 3. The method for producing a phosphorylated polysaccharide aqueous solution according to claim 1, wherein the functional group of the polysaccharide includes at least one selected from the group consisting of an alkyl group, a hydroxyl group, and a carbonyl group.

12. 3. The method for producing a phosphorylated polysaccharide aqueous solution according to claim 1 or 2, wherein the polysaccharide is a water-soluble polysaccharide in which a total of two or more pentoses and / or hexoses are linked by glycosidic bonds, and the functional group possessed by the polysaccharide includes at least one selected from an alkyl group, a hydroxyl group, and a carbonyl group.

13. 3. The method for producing a phosphorylated polysaccharide aqueous solution according to claim 1 or 2, wherein the polysaccharide comprises at least one selected from the group consisting of pullulan, dextran, starch, amylose, guar gum, glucomannan, hydroxypropyl cellulose, carboxymethyl cellulose, agarose, carrageenan, lactose, sucrose, sucralose, cellobiose, trehalose, maltose, isomaltulose, maltotriose, maltodextrin, cyclodextrin, glycosylsucrose, cycloamylose, glycogen, cluster dextrin, and lentinan.

14. the phosphate ester compound includes a compound represented by formula (P1), The method for producing a phosphorylated polysaccharide aqueous solution according to claim 1 , wherein the phosphate ester compound comprises a cyclic phosphate ester compound. 【Chemistry 2】 (In formula (P1), R １ , R ２ and R ３ are each independently a hydrogen atom or a substituent. １ and R ３ , R １ and R ２ , R ２ and R ３ , R ２ and R ２ may be bonded to each other to form a ring. １ and R ２ is a hydroxyl group, and R ３ is not a hydrogen atom, and n is an integer of 1 to 200.

15. 15. A method for producing a phosphorylated polysaccharide aqueous solution according to claim 1, 2 or 14, comprising reacting the mixed solution with a phosphate ester compound and then desalting the resulting reaction solution with an ultrafiltration membrane to remove by-products.

16. The method for producing an aqueous phosphorylated polysaccharide solution according to claim 15, wherein the purified product from which the by-products have been removed is subjected to cross-flow filtration using an ultrafiltration membrane to concentrate the phosphorylated polysaccharide to 0.15 to 10 wt %.

17. 15. A phosphorylated polysaccharide aqueous solution obtained by the method for producing a phosphorylated polysaccharide aqueous solution according to claim 1, 2 or 14.

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